聚氨酯泡沫固定产脂肪酶粗状假丝酵母(Candida valida T2)细胞的研究

对聚氨酯泡沫固定产脂肪酶粗状假丝酵母(Candida valida T2)细胞的固定化条件进行了研究.实验结果表明,聚氨酯泡沫颗粒经密度和粒径筛选,酸碱处理,以及固液比优化,载体固定细胞干质量比达到1571 mg/g,细胞脂肪酶的酶活为每克干细胞1415 U.电镜图片显示粗状假丝酵母菌(Candida valida T2)在载体孔隙内和脊壁上缠绕充盈,生长良好,固定结构稳定.固定化细胞连续催化水解桐籽油5批次,细胞相对水解酶活保持率达73%,固定细胞的损失率为12.5%.固定化细胞颗粒显示出良好的操作稳定性和酶活保持率,为进一步的应用研究提供了实验基础.     

D301树脂固定化假丝酵母脂肪酶

本研究选择7种吸附和离子交换树脂进行了假丝酵母脂肪酶(Candida sp.lipase)的固定化试验,通过测定固定化后各脂肪酶的酶活,筛选出固定化效果较好的弱碱性阴离子交换树脂D301;并通过扫描电镜将D301与脂肪酶Novozym 435的表面形貌做比较,进一步选定D301树脂作为载体,并对其采用戊二醛交联固定化,研究并优化了其固定化条件。结果表明,5%戊二醛溶液的加入量为8mL,处理时间为5h,酶液浓度为1.0g/L,磷酸缓冲盐溶液pH6.0,固定化处理10h效果最好,获得的固定化酶活力可达35U/mg,酶的固定化效率约为3.5U/(mg·h)。     

粗壮假丝酵母(Candida valida 20231)产脂肪酶发酵条件的研究

对粗壮假丝酵母(Candida valida 20231)产脂肪酶所需的培养基成分和发酵条件进行了优化。结果表明,粗壮假丝酵母在接种后3 h即进入对数期,18 h后转入稳定期,66 h后进入衰退期;发酵36 h达到最大酶活,至72 h仍然无明显下降趋势。最适合的培养基成分为:4.0%大豆粉,2.0%菜籽油,0.3%葡萄糖,0.3%NH_4NO_3,1.2%K_2HPO_4,0.42%NaH_2PO_4,0.4%MgSO_4·7H_2O。使用优化的培养基,最佳产酶的条件为:装液量80 mL(250 mL摇瓶),初始pH 6,接种量为2.0%,种子菌龄为12 h。在最佳产酶条件下,脂肪酶活力达到32.6 U/mL。     

毕赤酵母表面展示南极假丝酵母脂肪酶B全细胞催化合成葡萄糖月桂酸酯

利用表面展示南极假丝酵母脂肪酶B(Candida antarctica lipase B,CALB)的毕赤酵母细胞为全细胞催化剂,以葡萄糖为酰基受体,月桂酸为酰基供体,在非水相体系中催化合成糖酯。用硅胶柱层析对产物进行初提,再用制备液相色谱进一步分离纯化,并用高效液相色谱-质谱鉴定纯品性质。对该酶法合成糖脂反应体系进行了优化,其中考察了有机溶剂种类、复合溶剂体系中二甲基亚砜(DMSO)体积百分比、酶量、底物摩尔比、水活度和温度等几个影响酯化反应的因素。结果表明:在5mL反应体系中,以叔戊醇/二甲基亚砜(DMSO30%,V/V)为反应介质,添加初始水活度为0.11的全细胞催化剂0.5g,葡萄糖0.5mmol/L,月桂酸1.0mmol/L,60°C下反应72h后,葡萄糖月桂酸单酯的转化率达到48.7%。     

假丝酵母99-125脂肪酶喷雾干燥工艺及催化性质的研究

利用响应面法对假丝酵母脂肪酶喷雾干燥工艺条件进行优化,考察进口温度、雾化速度、保护剂含量对脂肪酶活力收率的影响。确定了最佳喷雾条件:保护剂为10～15 g/L的阿拉伯胶,进口温度115～120℃,雾化速度0.4 L/h,可得到收率最高为60.5%的脂肪酶酶粉。制得的固定化酶用于手性拆分(R,S)-1-苯乙醇,光学产率最高可达到53.6%;用于催化合成生物柴油,转化率最高可达到90.2%。在4、30℃下密封保存,半衰期可分别达到15个月、3个月。     

展示南极假丝酵母脂肪酶B的毕赤酵母全细胞催化合成短链芳香酯

展示酶的酵母细胞作为全细胞催化剂,既具有固定化酶的优点,又有制备简单、成本较低的特点。本研究将细胞表面展示南极假丝酵母脂肪酶B(Candida antarctica lipase B,CALB)的重组毕赤酵母用于非水相中催化合成短链芳香酯,通过滴定和气相色谱的方法测定底物酸的转化率,从底物的碳链长度、醇的结构、酵母冻干粉的添加量、底物浓度及底物的酸醇摩尔比等方面考察了展示CALB的毕赤酵母全细胞催化合成短链芳香酯的特性。研究结果表明:该全细胞催化剂可催化C10以下的酸和醇直接酯化合成多种短链芳香酯,酸的转化率达到90%以上;其中己酸和乙醇为酶的最适底物;酵母冻干粉的添加量20g/L(306.0U/g-drycell)、己酸浓度0.8mol/L、酸醇摩尔比1:1.1是合成己酸乙酯的最佳条件。在此条件下反应1.5h,己酸的转化率达到97.3%。在现有的关于脂肪酶非水相催化合成短链芳香酯的报道中,该全细胞催化剂显示出较好的底物耐受性以及较高的催化反应速率。因此,展示CALB的毕赤酵母全细胞催化剂在合成短链芳香酯方面具有较大的商业化应用潜能。     

南极假丝酵母脂肪酶A的表面展示及酶学性质研究

采用a凝集素作为载体蛋白,首次将南极假丝酵母脂肪酶A展示在酿酒酵母细胞表面,通过MD平板筛选获得表面展示型的CALA酵母工程菌株。免疫荧光检测显示CALA被成功展示在酵母细胞壁表面,重组子经诱导后能在三丁酸甘油酯板上形成透明圈,说明展示的CALA具有活性。重组酵母在液体培养基培养72 h,活性达到最高,为80.4 U/g干细胞。酿酒酵母展示的CALA最适温度及pH值为70°C和pH 8.0。经50°C保温2 h,仍含有60%水解酶活力。展示的CALA在pH 7.0和pH 8.0溶液中比较稳定。经DMSO处理2 h,展示的CALA仍保持70%的活性。以上结果表明酵母展示的CALA可作为一种有潜质商业用途的全细胞催化剂。     

南极假丝酵母脂肪酶B的修饰与改造

南极假丝酵母脂肪酶B是用途最为广泛的脂肪酶之一,可用于许多有机化合物、医药中间体等的合成.为了改善南极假丝酵母脂肪酶B的催化性能,提高其环境适应性,同时提高蛋白的表达量,降低生产成本,从而使其更适于工业化应用,许多针对酶蛋白分子的修饰及改造方法已在南极假丝酵母脂肪酶B中得到大量应用.本文从化学修饰、理性设计和非理性设计3个方面详细综述了南极假丝酵母脂肪酶B的修饰与改造的关键技术,包括定点突变、蛋白质融合、易错PCR和DNA改组等,同时也介绍了以上技术在改良南极假丝酵母脂肪酶B特性方面的诸多成功实例.     

南极假丝酵母脂肪酶B的酿酒酵母表面展示及其催化己酸乙酯的合成

从南极假丝酵母(Candida antarctica)基因组克隆得到南极假丝酵母脂肪酶B(Candida antarctica Lipase B, CALB)全基因片段, 利用连接肽celA Linker将CALB与酿酒酵母细胞表面展示蛋白a-凝集素的C端连接融合, 构建表面展示载体pICAS-celAL-CALB, 转化酵母后获得重组酵母菌Saccharomyces cerevisiae pICAS-celAL-CALB。该重组酵母菌经葡萄糖诱导表达及分析, 表明CALB已在酿酒酵母细胞表面成功展示, 水解活力达26.26 u/(g·dry cell)。重组酵母菌经冻干能有效地实现在非水相中全细胞催化己酸和乙醇酯化合成己酸乙酯。反应物己酸与乙醇的摩尔比为1:1.25, 己酸乙酯的产率为98.0%, 具有较好的操作稳定性。     

南极假丝酵母脂肪酶B的酿酒酵母表面展示及其催化己酸乙酯的合成

从南极假丝酵母(Candida antarctica)基因组克隆得到南极假丝酵母脂肪酶B(Candida antarctica Lipase B, CALB)全基因片段, 利用连接肽celA Linker将CALB与酿酒酵母细胞表面展示蛋白a-凝集素的C端连接融合, 构建表面展示载体pICAS-celAL-CALB, 转化酵母后获得重组酵母菌Saccharomyces cerevisiae pICAS-celAL-CALB。该重组酵母菌经葡萄糖诱导表达及分析, 表明CALB已在酿酒酵母细胞表面成功展示, 水解活力达26.26 u/(g·dry cell)。重组酵母菌经冻干能有效地实现在非水相中全细胞催化己酸和乙醇酯化合成己酸乙酯。反应物己酸与乙醇的摩尔比为1:1.25, 己酸乙酯的产率为98.0%, 具有较好的操作稳定性。     

Immobilization of Candida rugosa lipase on colloidal gas aphrons (CGAs)

A novel technique for immobilization of Candida rugosa lipase onto anionic colloidal gas aphrons (CGAs) is described. CGAs are spherical microbubbles (10-100 microm) composed of an inner gas core surrounded by a surfactant shell. In this initial study, greater than 80% lipase (w/w) was effectively retained on the CGAs. Leakage of protein from the CGAs and the activity of the adsorbed lipase decreased with increasing enzyme loading; this indicates that multilayers of lipase may be adsorbing onto the CGAs. The CGA-immobilised lipase displayed normal Michaelis-Menten dependence on substrate concentration and also exhibited greater activity than the free enzyme.     

Modelling the microenvironment of a lipase immobilized in polyurethane foams

The effects of partition of substrates and product on the modelling of the microenvironment of an immobilized lipase were evaluated using Response Surface Methodology. The esterification of butyric acid with ethanol in n-hexane, catalyzed by Candida rugosa lipase immobilized in two biocompatible and relatively hydrophilic polyurethane foams (“Hypol FHP 2002™” and “Hypol FHP 5000™”) was used as the model system. For each set of initial conditions, the final concentration of substrates and ethyl butyrate in the microenvironment, at equilibrium, Cmicro, were estimated by mass balancing bulk and foams. The Cmicro values obtained were used to estimate the corresponding partition coefficients of ethanol (P_EtOH), butyric acid (P_BA) and ester (P_EB), between the foams (microenvironment) and the bulk medium. Foams containing previously inactivated lipase, as well as lipase-free foams were used. For both substrates, Cmicro values were, in the majority of the experiments, higher than their macroenvironmental counterparts. The lowest Cmicro values were observed with the less hydrophilic foam (“FHP 5000”). A decrease of Cmicro_EtOH in both foams and Cmicro_BA in “FHP 5000” foams, was obtained upon lipase immobilization. P_EB values were, in all cases, close to zero. This is beneficial in terms of the shift in reaction equilibrium, product recovery and alleviation of product inhibition effects.     

Effects of isoproterenol on IL-2 and cAMP production in peripheral T cells from asthmatic and non-asthmatic subjects sensitive to Candida

Immunity to Candida albicans (Candida) is characterized by a Th-1 type pattern of reactivity. Candida is rarely a cause antigen for bronchial asthma. Beta agonists have been found to inhibit secretion of IL-2 from T cells through intracellular cAMP elevation. We examined effects of isoproterenol (ISO) on Candida-stimulated T cells. Peripheral T cells obtained from six Candida-sensitive asthmatics, six Candida-sensitive non-asthmatic subjects, and six normal donors by Ficoll-Hypaque gradient centrifugation and nylon-wool column chromatography were incubated with Candida antigen or concanavalin A (Con A) in the absence or presence of ISO. Secretion of IL-2 and intracellular accumulation of cAMP were assayed by ELISA. Con A induced secretion of IL-2 in each of the three groups. Candida antigen induced IL-2 secretion in the normal and the non-asthmatic subjects, but not in the asthmatics. ISO, which reduced Con A-induced secretion of IL-2 in a dose-dependent manner, had no effect on Candida-induced secretion of IL-2. Although ISO increased the intracellular concentration of cAMP in untreated and Con A-treated T cells from all donors, cells from the normal and the non-asthmatic subjects, but not from the asthmatics, that were co-incubated with ISO and Candida had lower levels of cAMP than those treated with ISO alone. It is suggested that Candida antigen induces secretion of IL-2 and reduces ISO-inducible accumulation of cAMP in Candida-responsive IL-2 secreting cells, which may make Candida-induced secretion of IL-2 insensitive to ISO.     

Kinetic Resolution of Profens by Enantioselective Esterification Catalyzed by Candida antarctica and Candida rugosa Lipases

Profens (2‐arylpropionic acids) are known as one of the major nonsteroidal antiinflammatory drugs (NSAIDs) used in the treatment of inflammation associated with tissue injury. The inflammatory activity of profens is mainly due to their (S)‐enantiomer, whereas they are commercially available not only as pure enantiomers, but as racemates as well. There are several methods widely used in order to obtain enantiomerically pure compounds, however, the kinetic resolution with the application of lipases as biocatalysts may have an added advantage in the production of optically pure active pharmaceutical ingredients, such as milder reaction conditions, reduced energy requirements, and production costs. The aim of this study was to compare the results described in the literature in the case of the influence of reaction medium, alcohol moiety, and reaction temperature on the catalytic activity of lipases from Candida antarctica and Candida rugosa. Chirality 26:663–669, 2014. © 2014 Wiley Periodicals, Inc.     

Immobilization of Candida rugosa lipase by cross-linking with glutaraldehyde followed by entrapment in alginate beads

Candida rugosa lipase was immobilized by first cross-linking with glutaraldehyde and then entrapping in calcium alginate beads. The presence of 2-propanol during cross-linking markedly improved the enzyme activity and activity recovery. Maximal enzyme activity (2.1?mmol?h^?1?g^?1 immobilized conjugate, wet weight) and activity recovery (117%) were observed at 30% (v/v) 2-propanol for hydrolysis of olive oil, which were 1.7 and 2.0 times higher than those of the immobilized enzyme prepared in the absence of 2-propanol. The half-life of the immobilized lipase prepared by entrapment after cross-linking in 30% 2-propanol was 1.6 times higher than that prepared by entrapment of the native lipase without cross-linking and 2-propanol pretreatment. The enantioselectivity of the former was 11 times higher than that of the latter for hydrolysis of racemic ketoprofen ethyl ester.     

Liquid-phase mass transfer in fixed-bed of polyurethane foam matrices containing immobilized anaerobic sludge

This paper reports on the results of liquid-phase mass transfer experiments carried-out in a bed of immobilized anaerobic sludge. The liquid superficial velocity (v_s) was found to deeply affect the liquid-phase volumetric mass transfer coefficient (k_sa) in the v_s range of 0.007 to 0.075 cm.s^–1. Moreover, k_sa increased exponentially with v_s in that range, due to the decrease of the external mass transfer resistance.     

聚氨酯泡沫半固定化培养盘基网柄菌

研究了聚氨酯泡沫应用于固定化盘基网柄菌的可行性,发现以简单处理过的聚氨酯泡沫为载体,能够高效实现盘基网柄菌的固定化培养。考察了载体粒径大小、载体量和摇床转速等对固定化培养的影响,在优化的培养条件和固定化条件下,盘基网柄菌的最大细胞密度是悬浮培养的2~4倍。